The proposed work deals with the continuing characterization of yeast mitochondrial DNA as well as studies on mitochondrial biogenesis using genetic, molecular and biochemical techniques. Additional nuclear and mitochondrial mutants that affect yeast mitochondrial function will be isolated and, together with those already isolated, will be characterized with respect to the nature of their lesions. Hopefully, some will be found that specifically inhibit mitochondrial macromolecular synthesis. One mitochondrially inherited mutant already is dated that codes for a temperature sensitive gene product, which in turn prevents yeast cell growth on non-fermentable substrate at non- permissive temperatures, will be studied in great detail. It now appears as though the mutation is not pleiotopic and from biochemical experiments points to an altered cytochrome oxidase. Thus, the mutation could be in one of the 3 polypeptide subunits that are components of cytochrome oxidase and that are coded for by mitochondrial DNA. Following the isolation of intact, covalently closed circular yeast mitochondral DNA (mtDNA), it will be used in in vitro experiments, in a coupled transcription/translation system, to study the polypeptides coded for by the mitochondrial genome. The intact mtDNA will be transcribed by E. coli RNA polymerase and the translated polypeptides detected and identified by immunoprecipitation. The effect of carbon source on the depression of yeast mitochondrial DNA will also be studied; these investigations will be coupled to those dealing with the interaction of mitochondrial genomes in zygotes during and following mating of haploid cells. In addition, the replication of mtDNA will be followed, making use of deoxybromouridylate as a label in yeast strains that incorporate dTMP and that are in a physiological state that specifically allows only the mtDNA to replicate.